新型基因载体巯基烷基化壳聚糖的制备及体外性质的初步研究

Preparation and characteristics of Thiolated N-alkylated chitosan for gene delivery in vitro

目的制备巯基烷基化壳聚糖(TACS)载基因纳米粒,并对其体外相关性质进行初步研究。方法复凝聚法制备TACS/pDNA复合纳米粒子,并用透射电镜对其的形态和粒径进行观察和表征;凝胶阻滞分析观察其对基因的保护情况;以Lipofectamine 2000转染试剂作为阳性对照,检测其对人胚胎肾细胞(HEK293)的转染活性;四噻氮唑盐(MTT)比色法测定其对细胞活性的影响。结果TACS/pDNA复合纳米粒子形态不很均一,粒径在390nm左右;凝胶阻滞分析证明了载体能有效地包裹和保护基因不受DNaseⅠ酶的消化;体外基因转染实验证实了TACS纳米粒子能够有效地转染HEK293,其转染效率远高于壳聚糖纳米粒子,略低于脂质体;MTT测定其对细胞活力影响甚微,对细胞的毒性明显低于脂质体。结论TACS有望成为一种有价值的新型基因载体。

Objective To prepare Thiolated N-alkylated chitosan（TACS） nanoparticles carrying gene and study its characteristics in vitro. Methods The TACS/pDNA nanoparticles were prepared by complex coacervation. The morphology of nanoparticles was irregular, observed by transmission electronic microscopy . The protection of the nanoparticles for pDNA was observed by gel electrophoresis. Transfcction activity of the nanoparticles was evaluated by gene transfection experiment in vitro in HEK293 cells. Lipofectamine 2000 vector was used as control. The effect of the TACS/pDNA nanoparticles on cell viability was illustrated with MTT assay. Results The average particle size was 390 nm determined by nanoparticle size analyzer. The TACS/pDNA nanoparticles could partially protect the encapsulated plasmid pDNA from nuclease degradation as shown by electrophoretic mobility analysis. The gene transfection experiment in vitro suggested that the nanoparticles could transfect HEK293 cells effectively and the transfection efficiency of the TACS/pDNA nanoparticles was much higher than that of chitosan/pDNA nanoparticles and lower than Lipofectamine 2000 vector. Conclusion TACS is a promising vector for gene delivery.